Ontogenic changes in the digestive enzyme patterns and characterization of proteases in Indian major carp Cirrhinus mrigala

Abstract

Digestive enzymes of Cirrhinus mrigala (Ham.) were studied during ontogenic development. Specific amylase activity was detected in first feeding fish. The enzyme activity decreased up to day-18 and then it increased with the age of fish to reach the highest level on day-34. Protease activity was 28.61 ± 8.90 mU mg protein−1 min−1 on day-4 and increased with the age throughout the study period. Trypsin activity was 31.86 ± 1.12 mU mg protein−1 min−1 on day-4. The activity decreased up to day-10 and from day-12 onwards increased up to day-26. Chymotrypsin activity was 14.56 ± 2.74 mU mg protein−1 min−1on day-4 and constantly increased up to day-26. A significant increase in lipase activity was observed between days-24 and 34. SDS-PAGE and substrate SDS-PAGE showed the diversity of protein (17.4–127.8 kDa) and protease activity bands (16.6–88.8 kDa) during ontogenesis. Soybean trypsin inhibitor, phenyl methyl sulphonyl fluoride, N-α-p-tosyl-l-lysine chloromethylketone and N-tosyl-l-phenylalanine chloromethylketone inhibited the protease activity up to 79.72–97.21, 65.55–94.83, 45.41–75.31 and 40.78–64.72%, respectively. Inhibition study in substrate SDS-PAGE revealed the abundance of serine proteases and the presence of isoforms of trypsin and chymotrypsin. Ethylenediamine-tetraacetate showed 5.56–22.78% inhibition of metal ion-specific enzyme activity.